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. 2022 Sep 23;298(11):102522. doi: 10.1016/j.jbc.2022.102522

Figure 1.

Figure 1

Generation of Δipcs−promastigotes. A, schematic of planned replacements for IPCS with drug markers for nourseothricin (SAT), G418 (NEO), and expression construct pXG-IPCS with blasticidin (BSD) resistance. Arrows represent the location of primers used for PCR; primers 1/4 and 3/2 tests for correct NEO 5′and 3′ replacements; primers 1/6 and 5/2 test for correct SAT 5′ and 3′ replacement; primers 7/2 tests for WT IPCS and 7/8 test for the pXG-born IPCS. Primer 1, SMB 2598; 2, SMB 2827; 3, SMB 2600; 4, SMB 2608; 5, SMB 2609; 6, SMB 2601; 7, SMB 2607; and 8, SMB 1421; sequences can be found in Table S1. B, PCR confirmation of parasite transfectants. IPCS::NEO replacement (lanes 1, 2, primers 1 + 4, expected size 860 bp; and 3 + 2, expected size 1024 bp); planned IPCS::SAT replacement (lanes 3, 4, primers 1 + 6, expected size 849 bp; and 5 + 2, expected size 933 bp); WT IPCS (lane 5, primers 7 + 2, expected size 882 bp); and pXG-BS-IPCS (lane 6, primers 7 + 8, expected size 124 bp). IPCS, inositol phosphorylceramide synthase.